The oncogenic Epstein-Barr virus (EBV) belongs to the family of gamma-herpesviruses and establishes lifelong persistent B cell infections in more than ninety percent of the human population (19, 20). In healthy individuals, the majority of EBV-infected B cells show limited viral gene expression and a resting phenotype. Terminal differentiation of latently infected cells into plasma cells leads to virus reactivation, production, and reinfection of B cells (21). Expression of all viral latency genes causes growth transformation and proliferation of infected B cells, which is reflected by the outgrowth of EBV-transformed lymphoblastoid B cell lines (LCL) in vitro and by the association of EBV with a variety of B cell lymphoproliferative diseases including different types of lymphoma in vivo (32). EBV infection is controlled by T cells as indicated by an increased incidence of EBV-associated malignancies in patients with congenital or iatrogenically induced T cell dysfunction (32), and by the successful treatment of EBV-associated post-transplant lymphoproliferative disease (PTLD) in hematopoietic stem cell transplant recipients by the infusion of polyclonal EBV-specific T cell lines (34, 35). Such lines are prepared by repeated stimulation of peripheral blood T cells with autologous LCL and contain CD4+ and CD8+ components. The antigens recognized by the EBV-specific CD8+ T cell component are mostly derived from latent as well as immediate early and early lytic cycle proteins (17). Although CD4+ T cell responses to these antigens have been detected in peripheral blood of EBV-positive donors, CD4+ T cells in LCL-stimulated preparations are almost exclusively directed against late lytic cycle antigens derived from structural proteins of the virus, which are efficiently presented on MHC II following CD21-mediated uptake of EBV particles by B cells (1, 2, 9, 24). Such T cells are cytolytic and able to prevent the proliferation of EBV-infected B cells and to inhibit the outgrowth of LCL from freshly infected B cells (2, 16). Targeting mostly non-overlapping sets of viral proteins and different phases of the virus' life cycle implies that CD4+ and CD8+ T cells complement each other in establishing protective immunity against EBV.
The successful treatment of immanent and manifest PTLD in hematopoietic stern cell transplant (HSCT) recipients by the infusion of EBV-specific T cell preparations has provided an important proof of principle for this form of immunotherapy, but owing to the considerable technical requirements and financial implications of extensive in vitro T cell culture, adoptive T cell therapy still has a limited role in the management of virus-associated complications in HSCT patients (28). Moreover, other EBV-associated malignancies, e.g. nasopharyngeal carcinoma and Hodgkin's disease respond less efficaciously to this form of immunotherapy, most likely because of immune evasion strategies by tumor cells such as non-expression of the EBNA3 family of proteins, the immunodominant targets of the latent antigen-specific CD8+ T cell response (12). To increase clinical efficacy of the T cell preparations and to implement this treatment modality as a conventional therapeutic option, generic and more direct approaches for the generation of EBV-specific T cell lines enriched in disease-relevant specificities need to be developed.
Two recent reports imply an important role of CD4+ T cells in establishing antiviral immunity. First, low endogenous CD4+ T cell numbers have been identified as important risk factors for the development of EBV-associated diseases in immunosuppressed patients (36). Second, patients with PTLD showed better clinical responses in a recent phase II trial when the infused T cell lines contained higher proportions of CD4+ T cells (15). For reasons unknown, the CD4/CD8 ratio in LCL-stimulated T cell preparations may vary from 2:98 to 98:3 (37).
U.S. Pat. No. 6,194,205 describes a method for the stimulation of T cells having a desired antigen specificity. Genes required for immortalization are introduced into antigen-presenting cells (APCs); said immortalising genes are preferably taken from EBV. Column 8 lines 31-32 states the introduction of all EBV immortalizing genes into the cells by P3HR1 virus. The immortalising genes of EBV are present for instance on mini-EBV vectors as described in column 4, lines 39 and 40. Said APCs may be e.g. B cells as described in column 4, line 19. In column 9, line 63 to column 10, line 56 the use of EBV-immortalised LCLs for T cell activation is described. The method described in this US patent is different from the presently claimed method as no virus-like particles of EBV are used. EB virus-like particles do not include any transforming DNA but only EBV structural antigens necessary for virion production. Therefore, the method described in U.S. Pat. No. 6,194,205 suffers from the same drawbacks as all other prior art methods directed to providing CD4+ T cells specific for EBV-structural antigens. No LCLs have to be generated in the present method. Summarizing, U.S. Pat. No. 6,194,205 reports the use of lymphoblastoid cell lines, or continuously growing B cells latently infected by a genetically modified virus as antigen-presenting cells. The viruses present in these cells retain replicative and transforming abilities. This experimental system therefore does not make use of viruses or of virus-like particles that are non-transforming. The present application presents a method that circumvents the use of LCLs the latter being less efficient and less safe than virus-like particles used in the present invention for the generation of EBV-specific CD4-positive T cells.
Moosmann et al: “Generation of EBV-specific T cells using a nonreplicating EBV vector” Immunobiology, vol. 210, no. 6-8, 2005, page 430, describe mini-EBV transformed B cell lines obtained by infection of B cells with EBV-like particles carrying the mini-EBV genome. Second sentence second paragraph states: “However, it encodes the full set of latent genes and can therefore mediate B cell transformation in vitro.” This document does also not overcome the prior art disadvantages by using mini-EBV genomes which are not devoid of any transforming viral DNA causing again danger of introducing transforming DNA into the patient. This document does not describe the use of EBV-like particles being devoid of any transforming viral DNA and containing only EBV-structural antigens.
Moosmann et al: “B cells immortalized by a mini-Epstein-Barr virus encoding a foreign antigen efficiently reactivate specific cytotoxic T cells.” Blood 1 Sep. 2002, vol. 100, no. 5, 1 Sep. 2002 (2002-09-01), pages 1755-1764, XP002469377 ISSN: 0006-4971 describe mini-EBV-immortalized B cell lines produced by infection of B cells with virus-like particles carrying mini-EBV DNA. The first paragraph of the abstract states: “Previously we constructed a mini-EBV plasmid that consists of less than half the EBV genome, is unable to cause virus production, but still immortalizes in vitro”. Only the method described by the present inventors use EB virus-like particles which are devoid of transforming EBV-DNA.
The preparations described in both documents of Moosmann et al. are profoundly different in nature from the virus-like particles and have opposite properties regarding the ability to transform normal B cells: They do not efficiently generate multiple CD4-positive EBV-specific T cell clones. As a matter of fact, these preparations are unable to stimulate immune responses against EBV structural antigens due to the genetic alterations that were introduced into the mini-EBV genome. They further contain large amounts of transforming viral DNA. The documents of Moosmann et al. and US '205 disclose so-called “virus-like particles” which are fully infectious EB viruses containing mini-EBV, a genetically modified genome that fully retains its transforming capabilities. Contrary thereto, the invention describes the use of mature intact EB virions devoid of transforming viral DNA.
Feederle R. et al: “Epstein-Barr virus BNRF 1 protein allows efficient transfer from the endosomal compartment to the nucleus of primary B lymphocytes.” Journal of Virology, October 2006, vol. 80, no. 19, October 2006 (2006-10), pages 9435-9443, XP002469378 ISSN: 0022-538 X describe the use of mini-LCLs which are primary B cells transformed by mini-EBV. The EBV-like particles disclosed therein are not devoid of any DNA but still contain the mini-EBV genome. One of the major drawbacks of the prior art, namely to avoid the introduction of transforming EBV-DNA into the patient, is not solved by Feederle et al. Only the present inventors have found that EB virus-like particles containing only structural proteins and no transforming DNA can be efficiently used to generate a preparation containing B cells presenting EBV-structural antigens in a method to prepare CD4+ T cells specific for EBV-structural antigens. The results referred to were obtained with a virus that lacks the BNRF1 gene. This BNRF1-null virus contains normal amounts of viral DNA. Additionally, the EB-VLPs of the invention lack the terminal repeats but preferably not BNRF1. The ability to produce virus-like particles (i.e. virions devoid of transforming viral DNA) is restricted to the viral mutant devoid of terminal repeats, an additional element not present in the BNRF1-null mutant described in Feederle et al.
Summarily, none of the above-discussed documents, neither taken alone nor in combination with each other, render the method of the present invention obvious. None of the methods described in the prior art documents disclose the use of Epstein-Barr virus-like particles which are devoid of transforming viral DNA and which contain EBV-structural antigens in a method to obtain a population of CD4+ T cells which is specific for the structural antigens of the Epstein-Barr virus and avoiding the problem of introducing transforming genes into the patient.
It is a problem of the present invention to provide a method for a simple, efficient and rapid preparation of T cell lines specific for EBV in order to receive a population of CD4+ T cells specific for structural antigens of EBV and free of any potentially contaminating EBV capable of infecting and transforming human B cells.